Adaptation is triggered at relatively low incident light
The principal photoprotective mechanism of MPB, alongside with the migration, consists of the thermal dissipation of energy through the xanthophyll cycle called non-photochemical quenching (NPQ). In diatoms, NPQ mechanism involves the de-epoxydation of the diadinoxanthin (Dd) to the diatoxanthin (Dt) [8], where the Dt biosynthesis can be linearly correlated with diatom NPQ [31–33]. The de-epoxidation state index (DES) increased significantly following the light exposure (Fig. 1, Van der Waerden test, p < 0.05) evidencing an intensification of NPQ and oxidative stress along this gradient. At medium light (ML; 250 µmol photons m-2 s-1 PAR), DES values were significantly higher than in Dark (D) and low light (LL; 50 and 100 µmol photons m-2 s-1 PAR) and significantly lower than in the higher light conditions indicating the initiation of NPQ in the MPB. Under the high light ‘s conditions (HL; 500, 750 and 1000 µmol m-2 s-1 PAR), DES were around 4 times higher than in dark (D) conditions. During the photosynthesis, reactive oxygen species (ROS) are formed as by-products, especially when the PS II reaction centers are getting overwhelmed under high irradiance exposures (Foyer, 2018). The ROS are highly reactive and can cause serious damage to the cell [34–36]. Therefore, photosynthetic organisms establish photo-protective mechanisms such as NPQ, and regulation of the light-harvesting pigment production and ROS detoxifying systems [37, 38]. Thus, the DES between Dd and Dt can be used as a proxy of the oxidative stress generation through photosynthesis in MPB organisms during light exposure. In this study, the high DES measured in HL showed that these light exposures triggered stress in MPB. Moreover, the values of DES observed in the 500 µmol m-2 s-1 PAR are similar to those found for the marine diatom Phaeodactylum tricornutum under the same light and time exposure [37]. The authors observed an initial response phase allowing the acclimatation of P. tricornutum to the light by involving the regulation of photosynthetic proteins’ genes, pigment metabolism and systems of ROS scavenging. Thus, at HL exposures, the diatoms and maybe other MPB’s organisms were likely in this initial response phase.
Metabolic responses to high light exposures show FAs’ central role in MPB light adaptation
To better understand metabolic changes under light exposure, a non-targeted metabolomic analysis was performed on the MPB samples and, together with pigment analysis, their fingerprints are presented in the BC-MFA (Fig. 2). The light exposure explained 44% of the total data variability (total inertia). The axis 1 discriminated the HL conditions from the LL and ML, corresponding to high and low DES conditions, respectively. However, in the BC-MFA (Fig. 1), D conditions are together with HL, showing that the MPB could engage in similar metabolic responses when light-deprived or under too strong irradiances. After data filtering, 35 compounds were found in CHCl3 fractions and 72 in MeOH fractions. The untargeted metabolomic analysis allows to extract and measure a wide variety of polar and semi-polar molecules. The BC-MFA analysis permitted to highlight the principal compounds affected by the light exposures which were annotated and gathered in the table 1.
The samples exposed to HL were essentially characterized by compounds from the CHCl3 fraction (Fig. 2 and Fig. 3). The 2-methyltricosane (C174), pristane (C28) and the hexadecane,2-methyl (C26) belong to the chemical family of alkanes. The alkene 5-octadecene (E) (C108) and an undetermined alkane (C110) were also produced in significantly higher proportion in HL than in the LL and ML conditions (Fig. 2, ANOVA, p < 0.05). The pristane can be formed by biotic and abiotic degradation pathways. It can be synthesized by anaerobic bacteria through the oxidation of chlorophyll phytyl side-chain or trimeric α-tocopherol originating from senescent phytoplanktonic cells [39, 40]. The significantly higher value of pristane in biofilm under HL and D (ANOVA, p < 0.05) could be thus linked to MPB organisms’ damage caused by the light stressing conditions. The 2-methyltricosane and the hexadecane,2-methyl are mono-branched alkanes. The latter are reported to be abundant in cyanobacteria [41, 42] and were proposed to be a biomarker of this group [43]. The significant variation of these two alkanes could thus be a response of biofilm cyanobacteria to the light stress. However, it would be surprising that this light stress response concerns only cyanobacteria since the studied microphytobenthic biofilm is dominated by diatoms [20].
Whereas the presence of alka(e)nes has been often measured in microorganisms, their physiological functions remain poorly known [44–46]. In cyanobacteria, they are suspected to play a role in cell’s growth and division, photosynthesis and salt tolerance [47–50] and their the presence in the thylakoïd membranes also regulates the redox balance under stress [50]. In microalgae, their functions are still not understood. They are presumed to be mainly present in membranes like other hydrocarbons and known to be biosynthesized from FAs by decarboxylation of the corresponding aldehydes [44, 51]. In terrestrial plants and algae, alkana(e)s are major constituents of waxes. They are known to protect them from stressing environmental conditions such as high light irradiances and ultraviolet exposures [52–56]. Moreover, the latter were shown to induce an increase of the branched-chain alkanes content in plants’ waxes [57]. Whereas it has been not investigated yet on marine cyanobacteria and microalgae, the alka(e)nes synthetized by these photosynthetic microorganisms probably also have similar photo protective properties.
In microalgae and various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus), C15–C17 n-alkanes biosynthesis are presumed to be linked to photosynthetic membranes where the decarboxylation is catalyzed by the recently described photoenzyme fatty acid photodecarboxylase (FAP), involving photons at each catalytic cycle [45, 58]. Thus, the significant increase of alka(e)nes, in the HL conditions could be explained by a higher FAP activity in the biofilm microalgae. The cyanobacterial alka(e)nes are synthesized by two distinct and mutually exclusive pathways which are not known as directly light-dependent [46, 59, 60]. The olefin synthase pathway which produces terminal olefins (1-alkenes) is not implicated in the synthesis of the alk(a)enes identified to vary in this study since none of them has the terminal olefins. Thus, it is more likely that, in cyanobacteria, the synthesis involved the fatty acyl-ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO) pathway. These results suggest that the FAAR /ADO pathway could be involved in the light-stress responses of cyanobacteria present in mudflat biofilms.
Under dark conditions, an alkanes accumulation was also observed in a strain of the marine phytoplankton Dicrateria rotunda (Harada et al., 2021). In our study, MPB’s organisms therefore could also have accumulated alka(e)nes under both stressing dark and HL conditions. The high values of alka(e)nes measured in the D are not linked with the light exposure and thus cannot be attributed to the FAP activity but rather to non-light-dependent pathways in response to oxidative stress generation. But it doesn’t exclude that FAP and non-light-dependent mechanisms could work simultaneously under HL exposures, leading to alka(e)nes’ synthesis in the microphytobenthic biofilm.
The HL conditions were also characterized by the fatty alcohol 1-tridecanol (C21) (Fig. 2, table 1). Because the marine bacteria are known to produce odd chains of fatty alcohols with branches (Roper, 2004), the significant higher values of 1-tridecanol in the 750 and 1000 PAR conditions compared to the LL and ML (ANOVA, p < 0.001) are probably linked with the bacterial or cyanobacterial compartment of biofilm. The fatty alcohol (Z)-hexadec-11-en-1-ol (M214) was also measured in significantly higher value in the 500 PAR conditions than in LL and ML conditions (Van der Waerden test, p < 0.05). The fatty alcohols are part of metabolism pathway concerning the alkanes and FAs, as well, since they can be formed from alkanes and be converted in FAs through oxidation processes (Ishige et al., 2003; Soltani et al., 2004). The significant increase of 1-tridecanol could thus be linked with the significant alkanes’ accumulation related to cyanobacteria described previously.
Table.1 Annotated compounds identified with the BC-MFA analysis to vary under light exposure (dimension 1, cos2 > 0.50 ; dimension 2, cos2 > 0.20)
Compound
|
|
Cos2
|
Rmatch
|
CAS
|
Formula
|
Name
|
RIlitt
|
RIexp
|
|
BC-MFA dimension 1
|
M111
|
0,69
|
916
|
1731-88-0
|
C14H28O2
|
Tridecanoic acid
|
1624
|
1608
|
C4
|
0,53
|
800
|
6308-98-1
|
C16H19N
|
N-phenylalanine
|
NA
|
NA
|
C21
|
0,57
|
878
|
112-70-9
|
C13H28O
|
1-Tridecanol
|
1577
|
1575
|
C56
|
0,72
|
738
|
544-64-9
|
C14H26O2
|
Myristoleic acid (C14:1-n5)
|
1783
|
1780
|
C57
|
0,67
|
635
|
5129-66-8
|
C16H32O2
|
Tetradecanoic acid, 12-methyl-
|
1788
|
1780
|
C59
|
0,87
|
914
|
7232-64-1
|
C16H32O2
|
Pentadecanoic acid
|
1820
|
1800
|
C108
|
0,69
|
885
|
7206-21-5
|
C18H36
|
5-Octadecene (E)
|
NA
|
1969
|
C110
|
0,55
|
NA
|
NA
|
NA
|
undetermined alkane
|
NA
|
1969
|
C125
|
0,65
|
NA
|
NA
|
NA
|
NA
|
NA
|
2013
|
C126
|
0,72
|
NA
|
NA
|
NA
|
NA
|
NA
|
2013
|
C174
|
0,55
|
785
|
1928-30-9
|
C24H50
|
2-Methyltricosane
|
2363
|
2364
|
C26
|
0,53
|
814
|
1560-92-5
|
C17H36
|
Hexadecane,2-methyl
|
1664
|
1667
|
C28
|
0,65
|
813
|
1921-70-6
|
C19H40
|
Pristane
|
1687
|
1676
|
Dd
|
0,71
|
|
|
|
Diadinoxanthin
|
|
|
Dn
|
0,67
|
|
|
|
Dinoxanthin
|
|
|
F.like2
|
0,57
|
|
|
|
Fucoxanthin like 2
|
|
|
|
BC-MFA dimension 2
|
M147
|
0,21
|
700
|
5129-58-8
|
C15H30O2
|
Tridecanoic acid, 12-methyl-
|
1686
|
1708
|
M214
|
0,38
|
784
|
56683-54-6
|
C16H32O
|
(Z)-Hexadec-11-en-1-ol
|
1867
|
1864
|
M289
|
0,33
|
820
|
56390-03-5
|
C15H36O5SI3
|
Methyl- alfa-Lyxofuranoside 3TMS
|
NA
|
1997
|
C6
|
0,46
|
918
|
629-59-4
|
C14H30
|
Tetradecane
|
1400
|
1378
|
C25
|
0,29
|
791
|
3892-00-0
|
C18H38
|
Pentadecane, 2,6,10-trimethyl- (norpristane)
|
1633
|
1629
|
C99
|
0,38
|
960
|
6386-38-5
|
C18H28O3
|
Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl) -4-hydroxy-
|
1943
|
1936
|
C178
|
0,35
|
890
|
2566-90-7
|
C23H34O2
|
Docosahexaenoic acid methyl ester (DHA)
|
2471
|
2431
|
Ca
|
0,37
|
|
|
|
Chlorophyll a
|
|
|
Ca.allo
|
0,36
|
|
|
|
Chlorophyll a allomer
|
|
|
Ca.epi
|
0,22
|
|
|
|
Chlorophyll a epimer
|
|
|
Dt
|
0,34
|
|
|
|
Diatoxanthin
|
|
|
F
|
0,30
|
|
|
|
Fucoxanthin
|
|
|
F.like1
|
0,35
|
|
|
|
Fucoxanthin like 1
|
|
|
F.like2
|
0,23
|
|
|
|
Fucoxanthin like 2
|
|
|
unknow.1
|
0,26
|
|
|
|
|
|
|
unknow.2
|
0,26
|
|
|
|
|
|
|
For GC-MS data (C, CHCl3 fraction and M, MeOH fractions), annotation was done with NIST 2017 (comp, compound; RI, Kovats retention indices; exp, experimental; litt, literature).
|
Lipids and FAs’ compositions of marine diatoms and cyanobacteria are known to play a role in their adaption under light stress, notably by changing the membranes’ characteristics with the increase in unsaturation of membrane lipids [61] and allowing the solubilization of Dd essential for its de-epoxidation into Dt in diatoms [62, 63]. But, to our knowledge, the roles of the alka(e)nes and fatty alcohols in response to light irradiance have not been investigated yet. The results of the present study confronted with literature knowledge emphasize that such molecules could protect MPB organisms from HL by regulating redox balance of the thylakoid membranes under oxidative stress generated by the overwhelmed photosynthesis. And their synthesis could be directly driven by the light exposure through the FAP activity or by FAs and fatty alcohols conversion through FAs’ oxidation processes. This shows FAs’ central role in MPB light adaptation. It has been previously shown that high light intensity, as well as blue and red wavelengths, increase the total lipids production of benthic diatoms and change their FAs profile [64, 65]. The influence of the light on FAs’ synthesis thus directly affects the alka(e)ne and fatty alcohol content in the biofilm organisms.
Metabolic fingerprints highlight diatom-bacteria interactions in light adaptation processes
All the FAs’ values found to be significantly affected by the light exposure (Fig. 4) were lower in the D and HL than in the LL and ML conditions, except for the Docosahexaenoic acid methyl ester (C22:6n-3, DHA; C178) (p < 0.05). The LL and ML conditions were characterized by 4 FAs (Fig. 2, table 1 and Fig. 4): the tridecanoic acid (M111) measured in the MeOH fraction, the myristoleic acid (C14:1n-5; C56), 12-methyltetradecanoic acid (anteiso-C15:0, 12-MTA; C57;) and pentadecanoic acid (C15:0; C59) measured in the CHCl3 fraction. The latter two were previously found by our research team in the same sampling location and by the same untargeted metabolomic analysis method [20]. They are known as heterotrophic bacteria markers, usually composed by odd carbon chains, iso- and anteiso-branched SFA and MUFA [66, 67] where the anteiso-C15:0 is generally associated with the genus Bacillus [68, 69]. The C14:1n-5, the anteiso-C15:0 and other branched-chain FAs are known to have antibacterial properties [70, 71]. The increase of these FAs under LL and ML could have impacted the bacterial community structure of the biofilm.
Although the diatoms dominate the MPB, most of the measured FAs which varied under light exposure seemed to have bacterial origin. In this work, the LL and ML conditions showed low DES values and they are in the range of optimal light levels for benthic diatoms [72, 73], the dominant compartment of the biofilm presently studied. The metabolites present in these conditions corresponded thus to a biofilm with photosynthetic organisms in an optimal physiological state. These changes could be attributed to the phototrophic bacteria or to heterotrophic bacteria in tight biological interaction with the autotrophs. Depending on the species, benthic diatoms can have different associated bacteria [74–76], and can also promote or disadvantage bacteria strains notably through the production of extracellular polymeric substances [77]. In intertidal mudflat biofilms, MPB and bacteria communities’ composition were observed to covariate, enlightening this strong biological interaction in situ [78–80]. In this study, the activity of MPB organisms under optimal photosynthesis state could have triggered metabolic responses of bacteria, enhancing, or inhibiting some of them. The light-induced stress can thus affect directly and indirectly the metabolisms and taxonomic structure of mudflat biofilm bacterial community.